A directed mutational approach demonstrates that a putative linoleate isomerase from Lactobacillus acidophilus does not hydrate or isomerize linoleic acid

Abstract

Catalytic promiscuity of enzymes is a field of general interest and the application of enzymes, catalyzing more than one chemical reaction has also industrial relevance. Furthermore, comparing the evolutionary relationship in protein families serves as basis for their engineering and adaption in practical applications. Oleate hydratases (EC 4.2.1.53) and linoleate isomerases (EC 5.2.1.5) act on related substrates and share substantial sequence similarity, but catalyze different transformations. The intrinsic cofactor FAD is found in both enzyme classes, but seems to perform different functions: for isomerases a redox-based mechanism was identified and for hydratases only a structural or charge stabilizing function was suggested. Several fatty acid converting reactions were found in Lactobacillus acidophilus including oleate hydratase and linoleate isomerase activity. Two genes that may encode these two enzymes were identified (GenBank AAV42528.1 and ABB43157.1), cloned, and the corresponding proteins were produced in E. coli, purified, and investigated. This revealed that only the oleate hydratase activity could be confirmed. As both enzymes differ by only three amino acids, a series of mutants was also created and investigated to shed light on their promiscuous behavior. Practical applications: Bacterial isomerization or hydration of cis-double bonds leading to the formation of trans-double bonds or hydroxyl groups within fatty acids has been recognized to be useful for biocatalysis, since no additional cofactor recycling is required despite the presence of the cofactor FAD.